1. Field of the Invention
The present invention relates to systems and methods for fluorescently imaging labeled two-dimensional samples. Particularly, this invention relates to such systems and methods applied to DNA microarrays and gels used in protein and other biological or chemical separation and/or purification processes, as well as other two-dimensional fluorescent or luminescent samples.
2. Description of the Related Art
Current fluorescent imaging systems, such as those most commonly employed in biotechnology, read only a limited number fluorescent labels simultaneously, e.g. typically two to four. In part, this fundamental limitation arises from the use of optical filters to separate the fluorescence of the different labels employed. Moreover, if a large number of different fluorescent labels were employed, performance would be very limited because all the labels would not have well separated fluorescent spectra; the spectra begin to overlap, and optical filters would no longer clearly resolve different label species under this condition.
The prior art optical filters are typically employed because they offer very good optical efficiency; losses at the desired wavelengths of light are quite small. Other spectroscopic techniques with better wavelength resolution generally have greater losses and lower throughput due to the optical devices employed (such as spectrometer slits, mirrors and gratings). This can significantly limit sensitivity and hence readout speed. Also, many prior art fluorescent imagers use lasers as fluorescence excitation sources, which the optical filter is chosen to block, minimizing scattered excitation light.
The technique of excitation emission matrix (EEM) fluorescence spectroscopy has been previously demonstrated by Warner et al. as described in Warner, I. M. et al., “Analysis of multicomponent fluorescence data,” Analytical Chemistry (1977), 49(4), 564-73 and Warner, I. M. et al., “Quantitative analyses of multicomponent fluorescence data by the methods of least squares and non-negative least sum of errors”, Analytical Chemistry (1977), 49(14), 2155-9. The authors showed that the composition of multicomponent mixtures can be quantitatively analyzed by the following approach. The excitation emission matrix of each known component is collected, i.e. its fluorescence intensity is measured as a function of both excitation and emission wavelengths, creating a data matrix. Similarly, the EEM of a mixture of said known components can be recorded, and its composition determined quantitatively by deconvolving the mixture EEM into its component EEMs via least squares or other programming techniques. This was successful even in cases of severe overlap and poor signal to noise ratio. However, the authors restricted their work to simple, homogenous samples and no spatial resolution was incorporated.
In addition, hyperspectral imaging allows the possibility of better spectral discrimination of fluorescent labels than the simple optical filters commonly employed in microarray labs-on-a-chip and similar sample fluorescence readers. However, hyperspectral imaging is distinct from excitation-emission matrices in that, while it separates the emission light spectrally, it does not require a multiplicity of excitation wavelengths and is typically performed with a single monochromatic excitation source. Thus, hyperspectral imaging lacks this extra spectral dimension. The imaging technique collects a data hypercube from a heterogenous sample comprising emitted light intensity as a function of position (x and y coordinates) and emission wavelength. Hyperspectral imaging has been applied to analysis of microarrays and microscopy in works such as O'Brien et al., “ASTRAL, a hyperspectral imaging DNA sequencer,” Reviews of Scientific Instruments (1998), 69(5), 2141-2146; Schultz et al., “Hyperspectral Imaging: A Novel Approach For Microscopic Analysis,” Cytometry (2001), 43, 239-247; and Martinez et al., “Identification and removal of contaminating fluorescence from commercial and in-house printed DNA microarrays,” Nucleic Acids Research (2003), 31(4) e18.
FIG. 1A is a block diagram of a conventional spectrofluorimeter 100. The spectrofluorimeter 100 employs a lamp 102 providing a broadband (e.g., white) light 104 delivered to an excitation monochromator 106 which filters the broadband light 104 to a near-monochromatic light 108. The monochromatic light 108 from the excitation monochromator 106 is directed to the test sample 110 where the incident monochromatic light output 108 causes the test sample 110 to fluoresce. The fluorescent light 112 emitted from the test sample 110 is directed to an emission monochromator 114 which filters the fluorescent light 112 to a specific emitted monochromatic light 116. If the specific emitted monochrome light 116 is present in the fluorescent light 112, it will be detected by the detector 118 indicating the presence and amount of the target species. By scanning over a multiplicity of excitation wavelengths for each or a multiplicity of emission wavelengths, an excitation-emission matrix (EEM) may be recorded. A variety of specific prior art devices are described hereafter.
FIG. 1B is a schematic diagram of a commercial excitation-emission matrix (EEM) instrument 120. The instrument 120 operates with excitation spectral light 122 having an wavelength variation along one dimension incident on a sample 124. The excitation spectral light 122 causes fluorescent emission which is then dispersed (shown schematically as a prism 126) along a dimension perpendicular to the spectral dimension of the excitation light 122. The dispersed fluorescent emission may be captured as an image 128 representing a two-dimensional array with the excitation wavelength along one axis and the emission wavelength along the other axis. A single sampling operation of the EEM instrument 120 yields information much more rapidly than a conventional spectrofluorimeter 100 as described above in FIG. 1A, i.e., an entire excitation-emission matrix is recorded in a single reading, without the need to scan either excitation or emission wavelength.
U.S. Pat. No. 6,323,944, issued Nov. 27, 2001, and U.S. Pat. No. 6,441,892, issued Aug. 27, 2002, both by Xiao, disclose a spectrofluorimeter employing a pair of linear variable spectral filters to produce a three dimensional data output (i.e., an EEM). A collimated white light source is used that first passes through a first linear variable spectral filter, then through a sample where fluorescence occurs, then the resultant light passes through a second linear variable spectral light filter that is oriented at ninety degrees from the first filter. The light is then detected by a CCD sensor for conversion into data. This arrangement provides a very simple, rugged and compact instrument that can be used almost anywhere, such as at the scene of a contamination accident.
U.S. Pat. No. 6,597,932 by Tian et al., issued Jul. 22, 2003, discloses an instrument for evaluating fluorescence of a heterogeneous tissue including means for exciting a two-dimensional portion of the tissue surface with excitation radiation at a plurality of excitation wavelengths, means for collecting emission radiation from the two-dimensional portion of the tissue surface simultaneously with excitation of the portion, and means for forming a two-dimensional excitation-emission map of the excitation radiation and the simultaneously collected emission radiation and spatially averaging the excitation and emission radiation. Note that the approach of Tian et al. performed a homogenization of the signal from the heterogeneous sample. Accordingly, it is not an imaging technique.
U.S. Pat. No. 5,459,325 by Hueton et al., issued Oct. 17, 1995, describes a high-speed fluorescence scanner for scanning a sample at equal angles. The scanner has most of its optical components, including a light beam source, a detector, and various filters, lenses, and reflectors, in a fixed position, removed from the scan head. The lightweight scan head contains a single reflector and lens combination which is reciprocated rapidly along one axis to lengthen and shorten a region of the path of a collimated excitation beam and to form a scan line on a sample. The fluorescence emission may be gathered by the lens of the scan head and directed back, generally along the optical path of the excitation beam, to a detector. Another embodiment of the scanner places the light source, in miniature form, directly on the scan head. The sample may be translated in an axis orthogonal to the scan line in order to stimulate fluorescent emission from a two-dimensional portion of the sample. The design of the optical assembly permits scan speeds of up to approximately 100 inches per second.
U.S. Pat. No. 6,211,989 by Wulfet al., issued Apr. 3, 2001, discloses a light scanning device for exciting and detecting secondary light, especially fluorescent light, on a sample, comprising a light emission device for emitting exciting light with a wavelength suitable for exciting secondary light on or in said sample, a focusing optics for focusing the exciting light on a sub-area of said sample, a sample holding device for releasably holding the sample, a detection unit comprising a detection optics for the secondary light emitted by the sample in response to excitation and a detector device for converting the detected and imaged secondary light into electric signals. In the case of conventional known light scanning devices, scanning is carried out by means of a deflection unit consisting of tilting mirrors. Due to the long path of the light beam, positioning inaccuracies of the tilting mirrors result in major position inaccuracies of the scanning ray bundle on the surface of the sample. For avoiding this disadvantage of the prior art, the light scanning device according to the invention makes use of a sample holding device which is adapted to be rotated for rotating the sample relative to the exciting light in such a way that different sub-areas of said sample can be excited by means of the exciting light so as to emit secondary light. Due to the mechanical rotary movement of the sample, a deflection of the scanning light beam relative to the optical axis is not necessary so that precise positioning of the scanning ray bundle on the sample is possible.
Also, U.S. Pat. No. 6,172,785 by Wulf, issued Jan. 9, 2001, discloses a light scanning device for exciting and detecting secondary light, especially fluorescent light, on a sample, comprising a light-emitting device for emitting excitation light having a wavelength which is suitable for exciting secondary light on or in the sample, a scanning unit for scanning at least one sub-area of the sample with said excitation light, and a detection unit for the secondary light emitted in response to excitation of the sample, said detection unit comprising a detection optics and a detector device. In the case of conventional scanning devices, the spatial resolution on the sample is determined by the scanning element alone. If spot detectors without spatial resolution are used, the detector must be read out and re-initialized after the illumination of each scanning spot on the sample; this results in a waiting time before the scanning beam can be moved to the next scanning spot and, consequently, in a reduction of the read-out velocity. For avoiding this drawback and for increasing the read-out velocity as well as for improving the spatial resolution on the sample, the device according to the invention makes use of a detector device comprising a large number of detection elements arranged in an array with predetermined position coordinates, said detection elements being arranged in an imaging plane of the detection optics and converting light detected in spatially resolved manner into electric signals.
U.S. Pat. No. 6,704,104 by Li, issued Mar. 9, 2004, discloses an array reader having a light source configured to emit an excitation light, a substrate comprising a plurality of sites spatially configured as a two-dimensional array having a plurality of rows and a plurality of columns, where each site is configured to support a sample. The array reader includes a changing device configured to determine which of said plurality of sites is illuminated at any given instant and a detector comprising a two-dimensional array of light sensitive elements, transmission grating beam splitter (TGBS) disposed along an optical path between the substrate and the detector, and a single light focusing element disposed along an optical path between the substrate and the detector. The TGBS is configured to receive non-collimated light emitted by at least one sample illuminated by said excitation light.
U.S. Pat. No. 5,631,734 by Stern et al., issued May 9, 1997, discloses fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.
U.S. Pat. Nos. 6,245,507 and 6,495,363, both by Bogdanov, issued Jun. 12, 2001 and Dec. 17, 2002, respectively. provide a hyperspectral imaging apparatus and methods for employing such an apparatus for multi-dye/base detection of a nucleic acid molecule coupled to a solid surface.
U.S. Pat. Nos. 6,373,568 and 6,690,466, both by Miller et al., issued Apr. 16, 2002 and Feb. 10, 2004, respectively, disclose an imaging system comprising an illuminator which produces illumination of any desired pure wavelength or of any selected mixture of pure wavelengths simultaneously, which illuminates a sample without spatio-spectral artifacts using illumination optics designed for that purpose, imaging optics, which form an image of the sample at a detector or viewing port, and a detector. This enables imaging the complete spectral image cube for a sample by taking sequential images while illuminating with a series of pure wavelengths, with greater ease and economy than by means of tunable filters, interferometers and the like. It further enables imaging while the sample is illuminated with a precisely controlled mixture of illuminant wavelengths, so that the image presented to the detector is a linear superposition of the sample properties at many wavelengths. This enables taking images of a sample that directly measure the weighted spectral properties such as projection pursuit vectors, principal components, and the like. Data acquisition is enormously simplified, and speed is increased by one to two orders of magnitude over existing techniques. This is of benefit in pathology, immunohistochemistry, Pap smear analysis, endoscopy, counterfeit detection, quality control, and other areas where one wishes to measure a spectral index of a living or inert sample.
U.S. Pat. No. 6,813,018 by Richman, issued Nov. 2, 2004, discloses a hyperspectral imager including a diffraction grating, a collecting reflecting element and a reimaging system. The diffraction grating has an entrance slit formed at an entrance slit location therein. The entrance slit has a long dimension oriented in a y-direction. The entrance slit transmits the radiation from a slice of an incoming scene image. The collecting reflecting element receives the transmitted radiation of the incoming scene image and reflects the transmitted radiation to a diffractive surface of the diffraction grating. Grooves on the diffractive surface are substantially parallel to the y-direction. The reimaging system receives radiation diffracted by the diffractive surface. The reimaging system produces a spectral image of the entrance slit at a focal surface. The spectral image provides a spectrum of radiation propagating through the entrance slit into the hyperspectral imager such that the spectrum of radiation from a first region in the y-direction can be distinguished from the spectra of radiation from other regions in the y-direction.
U.S. Pat. No. 6,427,126, by Dabiri et al., issued Jul. 30, 2002, discloses an advanced imaging spectrograph system and method are provided for very high throughput identification, sequencing and/or genotyping of DNA and other molecules. The system is based on the integration of improved electrophoresis structures with an imaging spectrophotometer that records the entire emission spectra along an imaging line across a sequencing gel (or capillary array). The system includes spectral shape matching to improve dye identification allowing the use of dyes having nearly any emission spectra and allowing greater than four dye multiplexing.
U.S. Pat. No. 6,495,818, by Mao, issued Dec. 17, 2002, discloses a microscopic hyperspectral imaging scanner that has a microscopic front objective lens, a spatial window for selectively passing a portion of the image therethrough, and a CCD array for receiving the passed portion of the image. The spatial window and CCD array are mounted for tandem reciprocating movement relative to the front object lens. In one embodiment, the spatial window is a slit and the CCD array is one-dimensional, and successive rows of the image in the focal plane of the front objective lens are passed to the CCD array by an image relay lens interposed between the slit and the CCD array. In another embodiment, the spatial window is a slit, the CCD array is two-dimensional, and a prism-grating-prism optical spectrometer is interposed between the slit and the CCD array so as to cause the scanned row to be split into a plurality of spectral separations onto the CCD array. In another embodiment, the CCD array is two-dimensional and the spatial window is a rectangular linear variable filter (“LVF”) window, so as to cause the scanned rows impinging on the LVF to be bandpass filtered into spectral components onto the CCD array through an image relay lens interposed between the LVF and the CCD array.
U.S. Pat. No. 6,490,075, by Scheps et al., issued Dec. 3, 2002, discloses an acousto-optic tunable filter hyperspectral imaging system which has applications that include detecting color variation in a region, for example, color variations due to temperature changes in an area of ocean water, and, in a more specific application, detecting bioluminescence of certain organisms known to attach themselves to various objects. In one aspect of the invention, an acousto-optic tunable filter hyperspectral imaging system is moved across the region to collect a series of images in which each image represents the intensity of light at a different wavelength. In one embodiment, the acousto-optic tunable filter hyperspectral imaging system includes a motion platform for positioning the acousto-optic tunable filter hyperspectral imaging system over successive Y-coordinates of a region in a direction substantially parallel to a direction of motion of the motion platform. In one such embodiment, the motion platform may be an aircraft or any other platform suitable for moving the acousto-optic tunable filter hyperspectral imaging system over the region.
U.S. Pat. No. 6,337,472, by Garner et al., issued Jan. 8, 2002, discloses a filter-less imaging microscope and method for analyzing samples on a slide at multiple wavelengths of light comprising, a microscope, a light dispersive element positioned to receive images from the microscope at multiple wavelengths, the light dispersive element producing an array of light from the image and a camera positioned to detect the light array produced by the light dispersive element, wherein the camera detects the light array dispersed by the light dispersive element at multiple wavelengths, is disclosed. The camera can detect the entire spectrum of light produced by the light dispersive element.
U.S. Pat. No. 5,379,065, by Cutts, issued Jan. 3, 1995, discloses a hyperspectral imager including a focal plane having an array of spaced image recording pixels receiving light from a scene moving relative to the focal plane in a longitudinal direction, the recording pixels being transportable at a controllable rate in the focal plane in the longitudinal direction, an electronic shutter for adjusting an exposure time of the focal plane, whereby recording pixels in an active area of the focal plane are removed therefrom and stored upon expiration of the exposure time, an electronic spectral filter for selecting a spectral band of light received by the focal plane from the scene during each exposure time and an electronic controller connected to the focal plane, to the electronic shutter and to the electronic spectral filter for controlling (a) the controllable rate at which the recording is transported in the longitudinal direction, (b) the exposure time and (c) the spectral band so as to record a selected portion of the scene through M spectral bands with a respective exposure time tq for each respective spectral band q.
In view of the foregoing, the prior art does not demonstrate a method for combining the advantages of the EEM in quantitative analysis of fluorescent species which have substantially overlapped spectra, with imaging capability necessary for spatially characterizing a sample consisting of a two-dimensional, heterogeneous array. There is also a need in the art for fluorescent imaging systems and methods that increase data throughput by the ability to handle a larger number of fluorescent species simultaneously while providing good quantitative accuracy and exhibiting reduced sample-to-sample variation. In addition, there is a need for such systems and methods to be cost effective. As detailed hereafter, these and other needs are satisfied by the present invention.